Unmanaged reward seeking is a shared central feature of eating and substance use disorders that expose patients to lifelong relapse vulnerability. Recent research shows that rewarding experiences induce synchronous activation of a discrete number of neurons in the nucleus accumbens core (NAcore) that are causally linked to reward-related contexts. This proposal intends to characterize the neuronal ensembles that are built through reward experience and code for reward seeking and extinction in the NAcore and adjacent circuitry. Throughout the proposal, special emphasis is given to comparing ensemble-specific changes between drugs (cocaine) and natural (sucrose) rewards to address whether or not addictive drugs usurp circuitry used by biological rewards or involve distinct circuitry mechanisms. I will use targeted recombination in active populations (TRAP) strategy, specifically the FosCreERxAi14 mouse line that allows tdTomato tagging of the neurons specifically activated during distinct behaviors. The K99 aims address the characterization of the cocaine- and sucrose-seeking ensembles and their colocalization within the same animal using an innovative version of the well-described model of reward self-administration and cue-induced reinstatement of seeking (Aim1A). The newly generated FosCreERxAi14xD2GFP reporter mice and RNAscope strategy (training component #1) will allow rigorous identification of the cell types comprising each ensemble (Aim 1B). Functional measurements of AMPA/NMDA ratios, a marker of plasticity (training component #2), will be performed within tagged cells during reward seeking (Aim 2). The R00 portion of the proposal evaluates necessity and sufficiency (Aims 3A-B) of the ensembles during cued-reinstatement using inhibitory and excitatory designer receptors exclusively activated by a designer drug (DREADD) specifically expressed in the ensemble cells, as well as the functional connection between reward-specific ensembles (Aim 3C). Characterization of the extinction ensemble will be achieved using FosCreERxAi14xD2GFP reporter mice and RNAscope (Aim 4A). Finally, ensemble-specific projections profiles of seeking and extinction will be completed using ensemble-dependent retroviral expression (Aim3B) followed by ensemble and projection specific plasticity measurements (Aim 4C). Preliminary data demonstrate that cocaine, sucrose and extinction ensembles form largely distinct but partly overlapping ensembles in the NAcore, and that AAV-targeted DREADD expression is restricted to the ensembles. The data obtained will shed new light on the mechanisms sustaining maladaptive reward-oriented seeking and extinction behaviors towards drugs and natural rewards. The Medical University of South Carolina provides an excellent research setting. Under the mentorship of Dr. Kalivas and my mentoring committee, I will experience advanced technical training and career development guidance to successfully complete this project while transitioning to an independent faculty position and further study of the intrinsic properties of ensembles underlying reward seeking.